It is well established that placental angiogenesis is essential for the increases in placental blood flow, which is directly correlate with fetal growth and survival and neonatal birth weights, and insufficient blood flows resulting in the shortage of oxygen and nutrient supply is associated with fetal intrauterine growth restriction and higher prenatal and neonatal morbidity or even mortality. Angiogenesis is regulated by a balance between angiogenic and anti-angiogenic factors. Placenta is the richest source of angiogenic growth factors, including VEGF, FGF, etc. Despite of the extensive studies on the involvement of angiogenic factors in placental angiogenesis, the precise molecular mechanisms regulating placental angiogenesis is still largely unknown. Recently, it has been shown that the neuronal guidance Slit/Robo signaling also possesses angiogenic activity in tumors, and blocking this signaling pathway inhibits tumor growth by suppressing angiogenesis. However, whether Slit/Robo signaling participates in the regulation of physiological angiogenesis like placental angiogenesis is unknown. In preliminary studies, we have detected the expression of the ligands, Slit proteins in both trophoblast cells and fetoplacental artery endothelial cells (EC), and receptors, Robo proteins in EC. The co- existence of Slit and Robo expression at the sites of angiogenic activity strongly suggest their role in placental angiogenesis. We hypothesize that this Slit/Robo signaling may play paracrine and autocrine role in regulation of placental EC angiogenesis, i.e. migration, differentiation and proliferation. The goal of this RO3 small grant application is thus to test this hypothesis through Specific Aim 1: to determine whether recombinant Slit proteins stimulate in vitro angiogenesis using the well-defined ovine fetoplacental artery EC (oFPAEC) model developed specifically for placental angiogenesis research;and Specific Aim 2: to determine whether trophoblast cells produce Slit proteins that in turn stimulate oFPAEC cell angiogenesis. Completion of this project will lay the foundation of our understanding of the regulation of placental angiogenesis by Slit/Robo signaling, and shed lights on the future studies of in-depth characterizations of the functional roles of this signaling in placental angiogenesis. The neuronal guidance Slit/Robo signaling cues have been recently shown to also regulate tumor angiogenesis and formation of vasculature during embryogenesis. In this RO3 proposal, we hypothesize that they participate in the regulation of physiological angiogenesis in the placenta, e.g. endothelial cell migration, differentiation and proliferation. The proposed studies will lay the foundation of our understanding of how neuronal guidance factors like Slit/Robo take part in the regulation of placenta function, especially on placental angiogenesis.